In most cell types, microtubules are organized by the centrosome, a specialized organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome matures as it gradually acquires increased levels of microtubule nucleating capacity and PCM. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. C. elegans is advantageous for such studies due to its suitability for cytological analysis and the ease with which it can be manipulated genetically.[unreadable] [unreadable] During 2006, we have continued our work on a newly identified set of genes that interact with a key regulator of centrosome duplication named ZYG-1. ZYG-1 is a kinase required for daughter centriole formation and in its absence bipolar spindle assembly fails producing an embryonic lethal phenotype. Previously, we described our identification and analysis of 40 independent suppressors that partially restore centrosome duplication and viability to animals carrying the temperature-sensitive zyg-1(it25) mutation. This year we have finished our initial genetic analysis of these suppressors and have determined that they represent 21 distinct szy (suppressor of zyg-1) genes. More importantly, we have made substantial progress in functionally characterizing three of them. [unreadable] [unreadable] One of these suppressors is a mutation in the sun-1 gene, which encodes an inner nuclear envelope protein. Together with ZYG-12, another nuclear envelope component, SUN-1 anchors the centrosome to the nucleus. Using RNAi, we have shown that loss of SUN-1, but not ZYG-12, can restore centrosome duplication to a zyg-1 mutant. Thus, SUN-1 opposes the activity of ZYG-1 in a ZYG-12 independent manner. Several other suppressors exhibit defects in attachment of the centrosome to the nucleus and thus it appears that we have identified a number of components of a new regulatory pathway. [unreadable] [unreadable] In addition to SUN-1, we have been studying the role of another factor identified in our suppressor screen. SZY-20 is a novel coiled-coil protein that localizes to the nucleolus, the nuclear periphery, and the centrosome. Loss of SZY-20 activity can partially suppress the centrosome duplication defect of a zyg-1 mutant. Likewise, we have found that szy-20 mutants can also restore centrosome duplication to a strain deficient in SPD-2, another key regulator of this process. szy-20 mutants also exhibit their own temperature-sensitive embryonic lethal phenotype. Interestingly, we find that szy-20 mutants possess enlarged centrosomes and that these centrosomes contain elevated levels of ZYG-1 and other proteins. We have performed both quantitative immunoblotting and quantitative immunofluorescence experiments and find that centrosomal components are expressed normally in szy-20 mutants. Thus, SZY-20 appears to antagonize recruitment of ZYG-1 and other factors to the centrosome thereby limiting centrosome size. This finding is of particular interest as SZY-20 is the first factor in any organism to be described with a role in limiting centrosome size. Finally we have also found that zyg-1(it25) can partially suppress the lethality of a szy-20(bs52) strain. The findings suggest a model wherein ZYG-1 and SZY-20 exist in a mutually antagonistic relationship. The molecular basis of this antagonism may involve competition for binding to a common factor. This yet-to-be-identified factor might be critical for the function of both ZYG-1 and SZY-20. [unreadable] [unreadable] We have also been studying a third ZYG-1-interacting factor named SZY-5. Loss of szy-5 activity produces a unique phenotype whereby tubulin assembles into large cytoplasmic aggregates in both embryos and germ cells. Interestingly, centriolar proteins co-localize with these aggregates, suggesting they represent abnormal intermediates in the centrosome duplication pathway. We have further found that this aggregation phenotype is sex specific; that is, aggregates only form in female germ cells but not male. This might be due to the high concentrations of centriolar proteins that are produced in the female germ line and stored in the oocyte for use in the embryonic cell divisions. We have cloned szy-5 and find it encodes a protein with four zinc fingers and a coiled-coil domain. Thus, SZY-5 might spatially and temporally regulate centrosome duplication by controlling the translation of one or more factors essential for this process. In the absence of szy-5, this factor could be inappropriately expressed causing the maternal stores of centriolar proteins to assemble into aggregates. We are currently working to determine the cellular distribution of SZY-5 and what factors it might be interacting with.[unreadable] [unreadable] Finally, my laboratory is also actively engaged in studying the process of centrosome maturation. Previously we uncovered a genetic interaction between zyg-1 and spd-5, a gene required for centrosome maturation. Cytological analysis of this interaction revealed an unexpected finding: ZYG-1 plays a role in the recruitment of PCM. Thus, ZYG-1 appears to function both in centrosome duplication and maturation. This result suggests the existence of mechanistic similarities between these two processes. We are currently working to determine what PCM components are recruited by ZYG-1 and if other proteins that function in centrosome duplication also function in maturation.